TNF-SP-EGFP融合蛋白的构建表达及亚细胞定位

Construction of TNF-SP Expression Vector with EGFP and Its Expression and Localization in COS-7 Cell Lines

目的探讨肿瘤坏死因子信号肽(TNF-SP)在哺乳动物细胞的表达和亚细胞定位。方法采用聚合酶链反应(PCR)技术,以TNF-SP-pcDNA3.0质粒为模板扩增TNF-SP基因,采用PCR产物的粘端克隆法,将TNF-SP定向克隆入pEGFP-N1的多克隆位点,构建TNF-SP-EGFP重组质粒,酶切,PCR检测,序列分析鉴定,采用脂质体转染法,将TNF-SP-EGFP融合基因转染COS-7细胞进行表达,经碘化丙啶(PI)染色后以激光共聚焦显微镜分析融合蛋白的表达及其亚细胞定位。结果PCR检测,酶切鉴定及测序证实目的基因TNF-SP正确连接到pEGFP-N1的多克隆位点。TNF-SP-EGFP重组体转染COS-7后,激光共聚焦显微镜显示TNF-SP-EGFP在细胞质表达。结论成功地构建了TNF-SP-EGFP融合蛋白真核表达质粒,在COS-7细胞中获得表达,并证实其定位于细胞质。

Objective To investigate the TNF-SP expression and its localization in mammalian cells. Methods TNF-SP gene was amplified with ploymerase chain reaction （PCR） from TNF-SP-pcDNA3.0 and was inserted into the eukaryotic expression vector pEGFP-N1. The recombinant plasmid TNF-SP-EGFP was identified with PCR, double enzyme digestion and DNA sequencing. The recombinant plasmid was transfected into COS-7 cell lines with lipofectamine. The transient expression and subcellular localization of the fusion proteins in the transfected COS-7 cells were analyzed by fluorescent microscopy and confocal microscopy. Results The recombinant plasmid TNF-SP-EGFP was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluorescence microscopy revealed that the TNF-SP-EGFP fusion protein was expressed in COS-7 cells 24 h after transfection. Confocal microscopy showed that the TNF-SP-EGFP was distributed in cytoplasm. Conclusion The recombinant plasmid TNF-SP-EGFP was successfully constructed and expressed in COS-7 cells. The fusion protein was located in cytoplasm.